Oscillatory signals regulate a wide variety of integral physiological and cellular processes, from G- protein coupled receptor (GPCR) signaling to circadian rhythms. Although an actively studied area, even the most well-known and commonly studied pathways can have controversy and lack of clarity on circuit architecture. This is because pathway perturbation studies using conventional molecular or genetic tools only provide limited information resulting in multiple plausible mechanisms. This proposal will develop tools and methods based on non-linear frequency and waveform response analysis to dissect such oscillatory pathways in ways that are not possible with conventional molecular or genetic perturbations alone. Specifically, we will use microfluidics to apply a periodic chemical input to cells and observed phase-locked cellular responses using real-time fluorescent readouts of intracellular signaling. The observed frequency response characteristics will be evaluated using computer models of the signaling pathway. Signaling circuit architecture as well as modes of action and mechanisms of inhibitors, agonists, and modulators will be dissected. Although the method should be applicable to any oscillatory signaling pathway, we will first focus on two GPCR signaling pathways (M3 muscarinic acetylcholine receptor and type 5 metabotropic glutamate receptor) that have very different proposed mechanisms of oscillation and that are physiologically and pharmacologically important (diabetes and schizophrenia). Aim 1. Analyze Phase Locking Response of Cells Under Base Conditions: Perform microfluidic pulsed stimulation of live cells with receptor ligands. Obtain high time resolution real-time imaging of intracellular signals using genetically encoded fluorescent indicators of calcium and IP3. Aim 2. Construct Mathematical Models of Signaling Circuitry: First construct plausible mathematical models based on published data. Then refine the circuit architecture and parameters to match observations in Aim 1, guided by results of uncertainty and sensitivity analyses. Aim 3. Delineate Mechanisms of Action of Modulators Through Phase Locking Analysis: Study how phase locking responses of cells change in the presence of inhibitors, agonists, and modulators. Use the experimental observations with mathematical models to delineate mechanisms of action. Aim 4. Disseminate self-regulating chips that make microfluidic phase-locking studies accessible to anyone.